2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polynuclear aromatic hydrocarbons are ubiquitous environmental contaminants with adverse effects on human health. These compounds cause toxicity by activating the aryl hydrocarbon receptor (AHR). The AHR also has physiological roles regulating vascular development, immune function, and cell growth, suggesting a role in human disease. To understand these diverse functions and the possible role of AHR in human disease, it is important to determine how AHR signaling is regulated. The negative regulation of AHR signaling is poorly understood. An inhibitor of AHR transcriptional activation function, AHR repressor (AHRR), has been identified, but its role in regulating AHR signaling remains enigmatic, and possible functions beyond the AHR pathway have been virtually ignored. Recent epidemiological studies have linked AHRR Pro185 and Ala185 polymorphisms to human reproductive disorders and AHRR has been identified as a likely tumor suppressor gene in humans. However, fundamental questions concerning the biochemical and functional characteristics of the AHRR and its variants remain unresolved, preventing a full understanding of its roles in human disease. The studies proposed here will utilize established vertebrate model systems (human cells and zebrafish embryos) to determine the transcription factor specificity and gene selectivity of AHRR and its polymorphic variants, the mechanism by which AHRR represses AHR and hypoxia inducible factors (HIFs), and the role of AHRR in regulating embryonic development and the response to TCDD and hypoxia in vivo. The central hypothesis is that AHRR acts through a transrepression mechanism to regulate the transcriptional activity of several transcription factors. In Aim 1, we will test the hypothesis that human AHRR can repress a variety of constitutively active and conditional transcription factors. We will also use gain-of-function (Tet-On) and loss-of- function (siRNA) experiments in human cell lines to determine the AHR and HIF target gene specificity of repression by AHRR. In Aim 2, we will use AHRR mutants, co-immunoprecipitation and chromatin immunoprecipitation assays, and ARNT-deficient cells to test several hypotheses: a) that the human AHRR and its variants act by a transrepression mechanism;b) that AHRR repression is ARNT-dependent;and c) that AHRR represses by binding to AHR and HIF or their transcription complexes. In Aim 3, we will investigate the in vivo function of AHRR in the powerful zebrafish embryo model system. We will generate germ-line null mutants for each of the two zebrafish AHRR paralogs. With these AHRR-null fish combined with AHRR overexpression experiments, we will assess the in vivo transcription factor and gene target specificity of AHRRs, the role of transrepression in the mechanism of action, and the roles of AHRR in embryonic development and the response to activators of AHR and HIF. PUBLIC HEALTH RELEVANCE: The aryl hydrocarbon receptor repressor (AHRR), a protein found in humans and other Vertebrate Animals functions to regulate the ability of toxicants such as dioxin and carcinogenic polycyclic aromatic hydrocarbons to cause altered gene expression and toxicity. AHRR polymorphisms have been linked to male infertility and endometriosis in women, and the AHRR has been proposed to function as a tumor suppressor gene. The proposed research will elucidate the mechanisms by which human AHRR and its polymorphic variants regulate the activity of signaling pathways involved in the response to hypoxia and toxicants such as dioxin, providing new insight into its role in human disease.